Abstract:
This thesis describes the investigation of quantum dot-doped particle fluorescent
technology commercially available for its application to analyte profiling in suspension.
The first part of the thesis described the characterisation of the quantum dot-encoded
microspheres, QDEMs, developed by Crystalplex (PA, USA). The multiple fluorescence
signatures of QDEMs were analysed using microscopy and flow cytometry technology
which provided high-content measurements with a single excitation sources and multiple
emission wavelength detectors. The sensitivity and stability of the materials was evaluated
under typical biomedical conditions encounter in multiple analyte suspension assays. Novel
analytical parameters were defined to study QDEM stability and confocal microscopy
detection system was used to provide structural and fluorescent imagines of the fluorescent
microspheres under various conditions. Composition of the aqueous environment,
temperature and physical forces applied to QDEM induced changes in their fluorescent
codes and structural properties. Optimal conditions were then defined for the application of
the material to biomedical assays. In a second stage, a conjugation method was developed
to produce optimised QDEM bioconjugates for the detection of single strand DNA in
suspension. The impact of the conjugation buffer, the concentration and the structure of
oligonucleotides was evaluated to optimise QDEM bioconjugates. Then, a novel approach
was investigated to optimise the hybridisation of ssDNA to QDEM bioconjugates.
Experimental design with response surface methodology determined optimum conditions
for the hybridisation of oligonucleotides to QDEM surface in suspension array. Finally, the
specific hybridisation of ssDNA to QDEM bioconjugates in a small liquid format adapted
to single nucleotide polymorphism detection was demonstrated.
The work presented here shows the potential of QDEM bioconjugates for suspension array
technology and DNA genotyping. Further, this report highlights the challenges that remain
for QDEM fluorescent technology to be reliable for biomedical and suspension array
applications.
